The present invention relates to the delivery of erythropoietin (EPO) to a mammal. More particularly, the present invention relates to provision of EPO in a mammal by means of expression from encoding nucleic acid included in an expression vector, that is by means of gene therapy. The present invention is based on the inventors"" experimental demonstration that therapeutic levels of EPO can be achieved using helper-dependent adenoviral (Hd-Ad) vectors, which levels are far beyond any levels previously attained using a variety of vectors, including adenoviral (Ad) vectors (i.e. non-helper-dependent).
Erythropoietin (EPO) is a protein of great interest because of its therapeutic usefulness in a variety of diseases. As is well known, the gene for human EPO was cloned by Amgen (see e.g. WO85/02610, EP-A-0148605) and recombinantly produced EPO (rEPO) has attained a huge market (in excess of 2.9 billion dollars). Currently, rEPO is administered to patients in protein form.
Despite its success, there is a number of problems with delivery of rEPO resulting in various unmet clinical needs, primarily because of the prohibitive cost of providing sufficient rEPO to achieve a long-term therapeutically effective dosage. Sufferers include individuals with anaemia of Chronic Renal Failure (CRF), anaemias due to beta-thalassaemia, and sickle cell anaemia (SCA). Large numbers of such individuals go untreated despite the fact that good therapeutic results can be achieved as long as enough EPO is provided.
In CRF there is an irreversible decline of kidney function, and the patients manifest a sequel of renal dysfunctions, including anaemia, but do not necessarily require dialysis except at the end stage renal disease (ESRD). At this stage the patient require either regular dialysis or kidney transplant.
CRF patients may be treated with rEPO, such treatment involving starting doses of 50-100 U/Kg, three times weekly, to achieve an increment of at least 5-6 point in the haematocrit. (Note that in vivo bioactivity of EPO is generally determined by the simple measurement of increase in haematocrit (Hct) by centrifugation of heparinsed blood in capillary tubes.) If this is not achieved within 8 weeks, the dose needs to be increased. Maintenance doses need to be individualised (Hct increase over 48%, are possibly deadly), with average of 75 U/Kg three times weekly, but ranging from 12.5 to 525 U/Kg three times weekly.
Circulating rEPO half life is of 4-13 hours if administered i.v., or 25-30 hours after s.c. administration. Over 95% of CRF patients respond well to the treatment, with a measurable Hct increase, and all are reported to become transfusion-independent after 2 months of treatment.
In both beta-thalassaemia and sickle cell anaemia the formation of a normal xcex12xcex22 haemoglobin (Hb) is impaired. Studies in baboons demonstrate that large doses (800-9,000 U/kg i.v.) of recombinant human EPO given i.v. increase gamma-globin chain synthesis and foetal Hb (AL-Khatt et al., N. En. J. Med., 1987, 317: 415-420).
Clinical trials with rEPO in SCA and beta-thalassaemia, with average doses of 500-1500 U/kg, show a rise in RBC count, Hb content and Hct. In SCA a significant increase of foetal Hb correlates with improved quality of RBC, reduced sickling episodes and improved quality of life.
There is no report of the insurgence of anti-EPO antibodies in CRF patients, even those treated for over 4 years, nor in patients with SCA or beta-thalassaemia.
What denies many individuals rEPO treatment is the costs of providing so much: xe2x89xa71,500 U/kg is required three times weekly for significant Hct increase and induction of xcex3-globulin synthesis in SCA patients.
The provision of an effective system for delivery of sufficient EPO would have ready application given the proven therapeutic effectiveness of the protein in diseases such as those discussed above.
The dosage of biologically active circulating EPO required to reach the therapeutic window for Hb-F stimulation is believed to be in excess of 0.900 U/ml (given that  greater than 1,5000 U/Kg three times weekly is necessary, and that the half life of EPO administered s.c. is 18 hrs). To date, despite many attempts using numerous approaches, there has been no report of such a level being achieved using a gene therapy approach, i.e. delivery of EPO by means of expression in the body from encoding nucleic acid conveyed within a recombinant vector.
Delivery of EPO cDNA by different means are described in the prior art. The highest level of circulating EPO reported is around 0.75 U/ml (Kessler P. D. et al. 1996, PNAS 93, 14082-7), well below the level required to have beneficial effect in SCA or beta-thalassaemia.
Studies have been published by other laboratories using Adeno vectors carrying the EPO gene which have shown limitations of these vectors in providing appropriate gene dosage and controlled hormonal release over a prolonged period of time.
Descamp et al., (1994, Human Gene Therapy 5:979-985) used an adenovector containing monkey EPO cDNA under the control of an RSV-LTR promoter. A minimum of 5xc3x97109 viral PFUs (plaque forming units) were required to give Hct increase (observed in a subset of animals). Example 15 below includes a comparison of results achieved using an embodiment of the present invention with results of Descamp et al.
Svensson et al. (1997, Human Gene Therapy 8:1797-1806) used an adenovector containing a mouse EPO coding sequence operably linked to an EF1xcex1 promoter, injecting particles i.m. Maximal EPO levels with 109 PFUs/mouse was 90 mU/m. No real dose response was shown. The same group earlier published Tripathy et al., 1996 Nat Med., 2: 545-50, and Tripathy et al., 1996 PNAS USA 93, 10876-80, in the latter of which naked DNA was injected into muscle, the maximal EPO levels achieved in blood being 50 mU/ml. The minimal amound of naked DNA needed to observed Hct increase was 10 mg/mouse, 500 mg/kg, corresponding to 35 mg/injection for a human of average 70 kg weight, a huge amount. Example 14 below includes comparison of results achieved using an embodiment of the present invention compared with the results of Svensson et al.
Remarkably, the experimental work described below demonstrates that use of embodiments of the present invention allows for circulating EPO levels of 300 U/ml to be achieved, i.e. far in excess of the level required for effective therapy. Furthermore, these levels can be obtained following a single injection and sustained over long periods of time.
The present invention in various aspects and embodiments employs an EPO encoding sequence within an adenovirus vector in which the entire Adenoviral genome coding sequences have been removed and substituted with exogenous DNA stuffer sequences, generally a Helper-Dependent Adenovirus vector (Hd-Ad).
A general aspect of the present invention provides for the use of such an adenoviral vector in delivery of erythropoietin to an individual. Such delivery is especially at a level in the serum of the individual of at least about 0.005, or 0.1, or 0.5, or 1, or 2, or 3, or 4, or 5, or 6, or 7, or 8, or 9, or 10 U/ml, and may be greater than about 10 U/ml, such as about 20, 30, 40 or 50 U/ml. As noted, levels of up to 300 U/ml are achievable using embodiments of the present invention.
One unit (U) of EPO corresponds approximately to 10 ng of pure protein and can be defined with reference to the international standard WHO-EPO 2nd International Reference Preparation (Annable et al., 1972, Bull. Wld, Hlth. Org. 47: 99) as the amount that is required to produce equivalent [3H]-thymidine incorporation into spleen cells from phenylhydrazine-treated mice to that expressed by 1 unit of the WHO standard preparation, or the amount needed to induce 50% of maximum growth (FC50) in erythroleukaemia cells, TF1.
One advantage that may be attained using the present invention is a dose response curve, making it possible to calculate the amount of vector to administer to achieve a desired level of circulating EPO.
One aspect of the present invention provides a method of delivering erythropoietin (EPO) to an individual, the method including provision in the individual of an adenoviral vector ad disclosed including nucleic acid encoding erythropoietin operably linked to regulatory sequences for production of erythropoietin in the individual by expression from the nucleic acid, especially whereby on such expression a dosage of erythropoietin at a level as indicated.
A further aspect of the present invention provides an adenoviral vector as disclosed including nucleic acid encoding erythropoietin operably linked to regulatory sequences for production of erythropoietin by expression from the nucleic acid. Such a vector may be provided for use in delivery of erythropoietin to an individual, especially for delivery of erythropoietin at a level in the serum of the individual as indicated.
A still further aspect of the present invention provides the use of an adenoviral vector as disclosed including nucleic acid encoding erythropoietin operably linked to regulatory sequences for production of erythropoietin by expression from the nucleic acid, in the manufacture of a medicament for delivery of erythropoietin to an individual, especially to the serum at a level as indicated.
A feature of embodiments of the present invention is the opportunity for repeat administration. Adenoviral vectors according to the invention may be re-administered to an individual to which such a vector (e.g. even of the same serotype) has previously been administered, e.g. at or more than about one month, e.g. 35 days, preferably at or more than about two months, more preferably at or more than about three months, more preferably at or more than about four months, more preferably at or more than about six months, more preferably at or more than about nine months, more preferably at or more than about 12 months, more preferably at or more than about 18 months, more preferably at or more than about 24 months from the first, or previous, administration. The re-administration may result in equivalent or greater levels of erythropoietin in the serum and/or increase in haematocrit.
The present invention allows for delivery of erythropoietin to achieve stable elevated levels in the serum.
For each aspect, either in addition to provision in the serum at at least the indicated level or alternatively, the provision may be for delivery of erythropoietin for an increase in the haematocrit reading of about 5%, or about 10%, or about 15%, or about 20%. The level of haematocrit may reach about 30%, or about 35%, or about 40%, or about 45%, or about 50%. For instance an increase of 10-15% on a reading of 30% for an anaemic level, i.e. to physiological levels of around 40-45%, may be achieved.
The level of erythropoietin and/or increase in haematocrit may be maintained to at least about 70%, more preferably at least about 80%, more preferably at least about 90%, more preferably at least about 95% of the level attained (that is attained following the first or previous administration) for at least about one month, preferably at least about two months, e.g. at least 84 days, more preferably at least about three months, more preferably at least about four months, e.g. at least 140 days, more preferably at least about six months, more preferably at least about nine months, more preferably at least about 12 months, more preferably at least about 18 months, more preferably at least about 24 months from the first, or previous, administration.
The individual may have a disease or disorder such that delivery of erythropoietin is of benefit or has a therapeutically beneficial effect. Delivery of erythropoietin may ameliorate one or more symptoms of the disease or disorder.
Diseases and disorders that may be treated in accordance with the present invention include anaemia of Chronic Renal Failure (CRF), anaemias due to beta-thalassaemia, and sickle cell anaemia (SCA), other anaemias including following radiation therapy or chemotherapy.
As indicated by the experiments included below, such high levels of expression may be achieved using embodiments of the present invention (e.g. using the EF1xcex1 promoter) that an excess of EPO may be delivered, which may result in side-effects in certain situations or individuals.
In SCA and in beta-thalassaemia it appears that excess production of EPO would not be detrimental, because even at very high EPO level, the maximum production of foetal-Hb is naturally limited and will not result in a dangerous increase of the Hct.
For delivery in other situations, e.g. for treatment of CRF patients, some more stringent regulation of gene expression may be employed, e.g. using a different promoter.
Parks et al. (1996). Proc. Natl. Acad. Sci. USA 93:13656-13570 describes a convenient packaging system for the large scale preparation of Helper-Dependent adenovirus vectors. In this system the Helper-Dependent vector is transfected in 293Cre cells, stably expressing the Cre recombinase. The cells are infected with the Helper Adenovirus, which furnishes in trans all the functions needed for the vector replication and packaging. The packaging signal of the Helper Adenovirus is flanked by two lox P sites, which are recognized by the Cre recombinase expressed in 293Cre cells: the packaging signal is thus removed and the majority of the Helper Adenovirus genomes cannot be packed, leading to preferential packaging of the Vector. After full cytopathic effect is obtained, the crude lysate is used to infect fresh 293Cre cells, coinfected with Helper Adenovirus: repeated cycles of infection bring to amplification of the vector. Roughly 20% of the Helper Adenovirus genomes escape Cre-mediated packaging signal excision, leading to the generation of a small amount of Helper Adenovirus. This small amount of contaminant can be easily removed by CsCl buoyant density centrifugation, leading to packaged helper-dependent vector preparations which are highly purified, with less that 0.1% helper Adenovirus contamination (Parks et al., 1996).
Suitable vectors may include the pSTK120 backbone employed by Morsy et al. (July 1998) PNAS USA 95: 7866-7871 in a HD-Ad vector for delivery of a leptin transgene to ob/ob mice.
The erythropoietin encoding sequence may be placed within the vector in any site that operably links it to the regulatory sequences for expression whereby the indicated level of erythropoietin and/or increase in haematocrit is achieved on expression in an individual. Suitable cloning sites are SWA I (NT 8096), EAG I (NT 16520), FSE I (NT 25653), SRf I (NT 14735), EcoRI (NT 24916, BspEI (NT 24321).
The regulatory sequences for expression will include a promoter.
By xe2x80x9cpromoterxe2x80x9d is meant a sequence of nucleotides from which transcription may be initiated of DNA operably linked downstream (i.e. in the 3xe2x80x2 direction on the sense strand of double-stranded DNA).
xe2x80x9cOperably linkedxe2x80x9d means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is xe2x80x9cunder transcriptional initiation regulationxe2x80x9d of the promoter.
Other regulatory sequences including terminator fragments, polyadenylation sequences, enhancer sequences, marker genes and other sequences may be included as appropriate, in accordance with the knowledge and practice of the ordinary person skilled in the art: see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucleic acid, for example in preparation of nucleic acid constructs, mutagenesis, sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Current Protocols in Molecular Biology, Ausubel et al. eds., John Wiley and Sons, 1992.
As noted, different promoters and other regulatory sequences may drive production of erythropoietin with different levels of promoter activity.
xe2x80x9cPromoter activityxe2x80x9d is used to refer to ability to initiate transcription. The level of promoter activity is quantifiable for instance by assessment of the amount of mRNA produced by transcription from the promoter or by assessment of the amount of protein product produced by translation of mRNA produced by transcription from the promoter, for instance by determining protein activity or evidence of protein activity (such as for erythropoietin an increase in haematocrit in an individual). The amount of a specific mRNA present in an expression system may be determined for example using specific oligonucleotides which are able to hybridise with the mRNA and which are labelled or may be used in a specific amplification reaction such as the polymerase chain reaction. Use of a reporter gene facilitates determination of promoter activity by reference to protein production.
EPO units (U/ml) may be assayed using the Quantikine IVD rhEpo ELISA kit of Accurate Chemical and Scientific, Minneapolis, Minn., USA, which is calibrated by the manufacturer against the international standard WHO-EPO 2nd International Reference Preparation (Annable et al., 1972, Bull. Wld, Hlth. Org. 47: 99).
Suitable promoters for use in aspects and embodiments of the present invention include the human elongation factor 1xcex1 (EF1xcex1) promoter, viral promoters such as CMV or RSV promoters, promoters linked to tet operator sequences, promoters linked to GAL4 binding sites, hypoxia inducible promoters, such as the Hypoxia Responsive Elements (HRE) naturally found in the erythropoietin gene.
Adenoviral particles containing nucleic acid encoding erythropoietin produced in accordance with the present invention may be formulated in pharmaceutical compositions. These compositions may comprise a pharmaceutically acceptable excipient, carrier, buffer, stabiliser or other materials well known to those skilled in the art. Such materials should be non-toxic and should not interfere with the efficacy of the active ingredient. The precise nature of the carrier or other material may depend on the route of administration, e.g. intravenous, cutaneous or subcutaneous, nasal, intramuscular, intraperitoneal routes.
Liquid pharmaceutical compositions generally include a liquid carrier such as water, petroleum, animal or vegetable oils, mineral oil or synthetic oil. Physiological saline solution, dextrose or other saccharide solution or glycols such as ethylene glycol, propylene glycol or polyethylene glycol may be included.
For intravenous, cutaneous or subcutaneous injection, or injection at the site of affliction, the active ingredient will be in the form of a parenterally acceptable aqueous solution which is pyrogen-free and has suitable pH, isotonicity and stability. Those of relevant skill in the art are well able to prepare suitable solutions using, for example, isotonic vehicles such as Sodium Chloride Injection, Ringer""s Injection, Lactated Ringer""s Injection. Preservatives, stabilisers, buffers, antioxidants and/or other additives may be included, as required.
Following production of adenoviral particles according to the invention and optional formulation of such particles into compositions, the particles may be administered to an individual, particularly human or other primate. Administration may be to another mammal, e.g. rodent such as mouse, rat or hamster, guinea pig, rabbit, sheep, goat, pig, horse, cow, donkey, dog or cat.
This may be for a therapeutic purpose, e.g. in delivery of a functional gene encoding an authentic biologically active product in a method of gene therapy, to treat a patient who is unable to synthesize that product or unable to synthesize it at the normal level or in normal form, thereby providing the effect provided by the wild-type and ameliorating one or more symptoms of the relevant disease.
Administration is preferably in a xe2x80x9cprophylactically effective amountxe2x80x9d or a xe2x80x9ctherapeutically effective amountxe2x80x9d (as the case may be, although prophylaxis may be considered therapy), this being sufficient to show benefit to the individual. The actual amount administered, and rate and time-course of administration, will depend on the nature and severity of what is being treated. Prescription of treatment, e.g. decisions on dosage etc, is within the responsibility of general practitioners and other medical doctors, or in a veterinary context a veterinarian, and typically takes account of the disorder to be treated, the condition of the individual patient, the site of delivery, the method of administration and other factors known to practitioners. Examples of the techniques and protocols mentioned above can be found in Remington""s Pharmaceutical Sciences, 16th edition, Osol, A. (ed), 1980.
The number of Adenovector particles to be administered in accordance with the present invention may be from around 1xc3x97103/g to about 1xc3x97106/g body weight, and may be about 1xc3x97103, or about 1xc3x97105, or about 1xc3x97106, preferably about 1xc3x97104/g body weight. Embodiments of the present invention allow for delivery of lower numbers of particles to be administered compared with work described in the prior art noted above, e.g. less than 1xc3x97109, more preferably less than 1xc3x97108.
A composition may be administered alone or in combination with other treatments, either simultaneously or sequentially dependent upon the condition to be treated. For example, in the case of SCA treatment may be in combination with hydroxyurea.
Delivery to a non-human mammal need not be for a therapeutic purpose, but may be for use in an experimental context. The erythropoietin for administration to a mammal of interest is preferably the erythropoietin of that species (including modified forms thereof) or a related species. Thus, for example, human EPO will generally be used for administration to humans (much as in the experiments described below murine EPO was administered to mice).
Further aspects and embodiments of the present invention will be apparent to those of ordinary skill in the art, in view of the above disclosure and following experimental exemplification, included by way of illustration and not limitation, and with reference to the attached figures.